Rapid detection of Candida albicans in clinical samples by DNA amplification of common regions from C. albicans-secreted aspartic proteinase genes.
Laboratory diagnosis based on genomic amplification methods such as PCR may provide an alternative and more sensitive method than conventional culture for the early detection of deep-seated candidiasis, an increasing cause of morbidity and mortality among immunocompromised patients. A novel method of DNA extraction from clinical samples based on treatment with proteinase K and isolation of DNA on a silica membrane was developed. The targets used for DNA amplification were the Candida albicans-secreted aspartic proteinase (SAP) genes, a multiple-gene family of at least seven members in C. albicans. A single pair of primers was designed in order to detect six of these SAP genes and, subsequently, to increase the sensitivity of the test. Detection of the PCR product by enzyme-linked immunosorbent assay was found to be as sensitive as Southern blotting with an SAP-labeled probe. The sensitivity of the assay was 1 cell/ml from serially diluted Candida cultures and 1 to 4 cells/ml from seeded blood specimens. The sensitivity and specificity of the present assay were tested in a retrospective study performed blindly with 156 clinical samples and were 100 and 98%, respectively, compared with the results of culture. For the subset of blood culture samples (n = 124), the sensitivity and the specificity were 100%. The two false-positive PCR samples came from patients treated with azole antifungal agents, indicating that PCR was probably able to detect damaged organisms that could not be recovered by culture.
Flahaut
M
M
Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. Flahaut@chuv.hospvd.ch
Sanglard
D
D
Monod
M
M
Bille
J
J
Rossier
M
M
D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000276:immunology; D002176:Candida albicans / Q000235:genetics*; D002177:Candidiasis / Q000175:diagnosis*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification*; D019286:Endopeptidase K / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005800:Genes, Fungal; D006801:Humans; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide
D017931:DNA Primers; D004271:DNA, Fungal; D012822:Silicon Dioxide; D019286:Endopeptidase K; D016282:Aspartic Acid Endopeptidases